Neurotransmitter release is acutely triggered by Ca2+ and is regulated during presynaptic plasticity processes that underlie some forms of information processing in the brain. Characterization of the mechanisms of release and its regulation is thus critical to understand brain function and will facilitate the development of therapies for neurological disorders with a presynaptic origin. Several proteins with crucial roles in neurotransmitter release contain multiple C2 domains, which are widespread Ca2+ and phospholipids binding modules but can also exhibit Ca2+-independent activities. These proteins include: i) synaptotagmin-1, the Ca2+ sensor that triggers fast release; ii) other synaptotagmin isoforms, which act as alternate Ca2+ sensors in different regions of the central nervous system and neuroendochrine cells, and modulate the Ca2+ sensitivity of release; iii) Munc13-1 and related isoforms, which are essential for synaptic vesicle priming and mediates diverse forms of presynaptic plasticity; iv) RIMs, which are Rab3 effectors that also have key roles in vesicle priming and presynaptic plasticity. The C2 domains of all these proteins are highly conserved and are hypothesizes in this proposal to regulate neurotransmitter release at multiple levels through their Ca2+-dependent and Ca2+- independent interactions. To test this hypothesis and gain insight into the diverse functions of C2 domains from these proteins in release, this application proposes studies of their structures and interactions by diverse biophysical methods, including NMR spectroscopy, X-ray crystallography, cryo-electron microscopy and fluorescence spectroscopy. This research forms part of an integrated approach where the biophysical data are correlated with genetic and functional experiments performed in the laboratories of close collaborators. Three Specific Aims are proposed. Aim 1 will continue ongoing studies directed at elucidating how synaptotagmin-1 triggers membrane fusion and neurotransmitter release in a Ca2+-dependent manner together with SNARE proteins and in a tight interplay with complexins, by characterizing their interactions. A particular focus will be placed at elucidating the structure of a quaternary complex formed by SNAREs, synaptotagmin-1, Ca2+ and phospholipids, which most likely plays a central role inCa2+-dependent membrane fusion. Aim 2 will continue studies devoted to compare the biochemical properties of other synaptotagmin isoforms involved in Ca2+- evoked exocytosis, and at deciphering the sequence determinants that underlie differences in these properties. These studies will shed light on the basis for functional differentiation between Syts, which is likely fundamental for brain function. Aim 3 will test the hypothesis that the Munc13-1 C2 domains control diverse forms of presynaptic plasticity through intramolecular interactions with the C-terminal MUN domain that plays a crucial role in vesicle priming, by characterizing these interactions and how they influence MUN domain activity. Potential interaction of RIM C2 domains that may underlie roles in vesicle priming and presynaptic plasticity will also be investigated.